DESCRIPTION: Well-regulated inflammation is brief in duration, characterized by rapid tissue infiltration and directed migration to sites of infection where neutrophils eradicate contaminating microbes, clear damaged tissue and initiate healing. When the response is excessive or prolonged, such as in patients with severe sepsis, neutrophils can damage the tissues they are recruited to protect, leading to organ failure and threatening survival. Mechanisms that regulate neutrophil function have largely been determined in vitro using two- dimensional glass or plastic surfaces that do not mimic the elastic, three-dimensional space of bodily tissues thereby diminishing the physiological relevance of those materials. As a result there is a significant gap in our understanding of how neutrophils function within the tissue microenvironment and how normal functions become abnormal in hyperinflammatory diseases. To begin to fill that gap, we studied neutrophils in vitro on pliable polyacrylamide gels that were conjugated with fibronectin and tuned to reflect the range of stiffnesses inherent among different tissues throughout the body. We reported that neutrophils are mechanosensitive as migration kinetics and generation of traction forces changed according to substrate stiffness. Neutrophils require integrins for adhesion and migration on two-dimensional substrates. However, following entry into the highly confined three-dimensional interstitial space, or in a 3D collagen gel, integrins become dispensable for migration. Therefore biomaterials that are designed to mimic the tissue environment must support integrin-independent neutrophil migration to be physiologically relevant. We engineered a double hydrogel sandwich system and used 3D traction force microscopy with new algorithms to track migrating neutrophils and measure spatiotemporal traction forces at high resolution. We show that migration and traction force generation are integrin-independent when cells are confined and, therefore, that the double hydrogel system is a biomimetic. We also show in this application that our 3D-TFM algorithms can be applied to acquire high-resolution maps of principal strains produced by neutrophils within 3D collagen gels. We show that neutrophils obtained from septic patients exhibit disorganized and excessive traction. Work in Aim 1 proposes to maximize the use of the double hydrogel system to map traction and migration of normal and activated neutrophils exposed to variations in matrix, confinement and substrate stiffness. Molecular mechanisms (actin polymerization; myosin contractility) that drive neutrophil motility under confinement will be determined. Aim 2 will embed cells within 3D collagen gels to determine motility and map principal strains during chemotaxis. Aim 3 will be performed in collaboration with physicians who manage septic patients in the Trauma ICU. We will show that dysregulated neutrophil traction and motility is an underlying molecular mechanism of the pathology caused by sepsis.